Sialomucin Complex (Muc4) Expression in Porcine Endometrium During the Oestrous Cycle and Early Pregnancy

The non‐invasive type of implantation in the pig is characterized by the maintenance of a thick glycocalyx coating on the uterine epithelial surface microvilli. Present study investigated the alteration in the sialomucin complex (Muc4) expression during the oestrous cycle and early pregnancy in the pig. Endometrial tissue samples were immunostained with the primary antibody to the Muc4 transmembrane subunit ASGP‐2. Muc4 immunostaining increased in the surface and glandular epithelia between days 5 and 10 of oestrous cycle. Immunostaining continued to increase on day 12 with the greatest intensity of uterine Muc4 immunostaining detected on day 15 of the oestrous cycle and early pregnancy. Endometrial Muc4 expression in cyclic gilts decreased dramatically during early proestrous but continued to remain abundant in the surface and glandular epithelium of pregnant gilts during the period of conceptus attachment to the uterine surface.     

Ovarian Cyclicity and Reproductive Performance of Holstein Cows Carrying the Mutation of Complex Vertebral Malformation in Japan

This study was carried out on 71 lactating Holstein Friesian cows to investigate the resumption of ovarian cyclicity postpartum and the reproductive performance in cows carrying the mutation of complex vertebral malformation (CVM) compared with control ones. The cows were distributed in two dairy farms in Hiroshima Prefecture, Western Japan. Blood samples were collected from the cows to detect carrier cows with CVM mutation. Furthermore, plasma samples were collected weekly after calving from control cows (n = 10) and CVM carrier cows (n = 10), until 10 weeks postpartum to investigate the day of first ovulation and the resumption of ovarian cyclicity postpartum. The reproductive parameters were investigated and compared with control and CVM carrier cows. Thirty‐six cows were diagnosed to be CVM carriers by DNA examination and confirmed later by DNA sequencing. The pedigree analysis of the carrier cows revealed that they were daughters of six types of CVM carrier semen that still was used in dairy farms in Western Japan. In terms of reproductive indices, there were no significant differences between the control and the CVM carrier cows on the day of the first ovulation postpartum and the interval from calving to first insemination. However, CVM carrier cows significantly required more inseminations per conception and showed a significantly longer period to conception and subsequent calving than control ones. In conclusion, the reproductive performance of the CVM carrier cows was lowered through conception failure that might indicate the occurrence of intra‐uterine mortality in those cows.     

Endometrial Phospholipase A2 Activity During the Oestrous Cycle and Early Pregnancy in Mares

The aim of this study was to determine phospholipase A2 (PLA2) kinetics and activity in the mare’s endometrium during the oestrous cycle and early pregnancy. Phospholipase A2 is responsible for the liberation of arachidonic acid from phospholipids, which is the first limiting step in prostaglandins synthesis. Phospholipase A2 activity was measured using an assay based on the liberation of oleic acid from 1‐palmitoyl‐2‐[¹⁴C] oleoyl phosphatidylcholine. The enzyme was shown to be calcium dependent, to have an optimum pH of 8 and an apparent Michaelis constant of 127 μm . Enzyme activity was low in the endometrium of early luteal phase tissue but increased significantly (p < 0.001) during the late luteal phase (5.39 ± 0.16; 3.48 ± 0.33, 6.85 ± 0.59, and 9.96 thinsp;± thinsp;1.23 thinsp;nmol oleic acid released/mg protein at oestrus, and Days 3, 8 and 14 after ovulation, respectively). The mean PLA2 activity in endometrial tissue from pregnant mares (4.23 ± 0.74) was significantly lower (p < 0.01) than from cyclic animals during late dioestrus (9.96 ± 1.23). The results indicate that PLA2 activity in equine endometrium changes with the stage of the oestrous cycle and thus may be influenced by systemic hormone concentrations. The inhibitory effects of conceptus products on secretion of prostaglandin during early pregnancy were associated with a competitive inhibitor that decreased endometrial PLA2 activity.     

Size Distribution of Dispersed Luteal Cells During Oestrous Cycle in Angora Goats

The present study examines the size distribution of the goat steroidogenic luteal cells throughout the oestrous cycle. Corpora lutea (CL) were collected after laparatomy on days 5, 10 and 16 of the oestrous cycle. Luteal cells were isolated from CL by collagenase digestion. Steriodogenic luteal cells were identified by staining of the cells for 3beta-hydroxysteroid dehydrogenase activity, a marker for steroidogenic cells. Luteal cells having steroidogenic capacity covered a wide spectrum of sizes, ranging from 5 to 37.5 microm in diameter. There was a significant increase in mean cell diameters (p < 0.01) as CL aged. The mean cell diameter on day 5 was 11.55 +/- 0.12 microm, which was significantly increased and reached up to 19.18 +/- 0.24 mum by day 16 of the oestrous cycle. The ratio of large to small luteal cells was 0.06:1.0 on day 5 of the oestrous cycle. This ratio increased to 0.78:1.0 by day 16 of the oestrous cycle. Luteal cell size on days 5, 10 and 16 of the oestrous cycle reached its maximum at 7.5, 10 and 35 microm in diameter, respectively. Development of CL is associated with an increase in luteal cell size in goats. It is likely that small luteal cells could develop into large luteal cells as CL becomes older during oestrous cycle in goats.     

我国荷斯坦种公牛CVM遗传缺陷基因的分子检测

研究运用引入酶切位点聚合酶链式反应(PIRA-PCR)方法检测牛脊柱畸形综合症(CVM)在我国荷斯坦种公牛中的携带情况。共检测了来自全国14个公牛站的587头种公牛,结果表明:携带者56头,携带率为9.54%。系谱分析确认40头携带者公牛是Carlin-M Ivanhoe Bell的后代。对公牛信息统计显示,2001—2007年各年度出生的种公牛都保持较高的CVM携带率(8.22%～15.56%)。美国、加拿大和澳大利亚是我国引进公牛的主要来源国,进口公牛的CVM携带率分别为11.37%、3.73%和14.29%。此外,本研究还对我国荷斯坦牛CVM遗传缺陷的控制和CVM携带公牛的利用提出建议。     

Leptin Gene and Protein Expression in the Ovary During the Oestrous Cycle and Early Pregnancy in Pigs

Leptin, the product of the obese gene, is the hormone originally identified in adipocytes. It is involved in the control of satiety and energy metabolism. More recent observations suggest that leptin plays an important role in reproduction. Leptin mRNA and protein have been found in the human and the murine ovary. However, the expression of leptin in the porcine ovary has not been examined. Therefore, the aim of the present work was to compare the expression levels of porcine leptin mRNA by semiquantitative RT‐PCR and in situ hybridization, as well as leptin protein by Western blotting in the corpus luteum (CL) and ovarian stroma (OS) during mid‐ and late‐luteal phase of the oestrous cycle as well as during days 14–16 and 30–32 of pregnancy. Leptin gene and protein expression in CL was increased on days 14–16 of the cycle compared with pregnant animals. Leptin gene expression in OS was higher during the late‐luteal phase of the cycle than on days 30–32 after conception. However, comparison of leptin protein expression in OS between days 14–16 of the cycle and days 30–32 of pregnancy indicates a higher protein expression during pregnancy. Moreover, leptin gene expression was higher in porcine CL and OS on days 14–16 of pregnancy in comparison to days 30–32. Contrary to leptin mRNA expression, a higher leptin protein expression was observed on days 30–32 compared with days 14–16 after conception. In summary, the present study provides the first evidence that leptin mRNA and protein occur in porcine ovary and vary during the oestrous cycle and pregnancy. Moreover, the obtained results indicate that also locally synthesized leptin may participate in the control of pig reproduction by exercising its action at the ovarian level.     

北京地区荷斯坦牛脊椎畸形综合征的调查研究

脊椎畸形综合征（Complex Vertebral Malformation,CVM）是由常染色体上SLC35A3基因单碱基突变（G→T）引起的隐性遗传疾病,该基因隐性纯合（CV/CV）时奶牛致死,但CVM携带者表现正常,所以CVM携带者公牛可以通过人工授精技术传播CVM缺陷基因。本研究利用PCR-SSCP方法对北京地区242头公牛样品和403头母牛样品进行了检测分析,研究结果表明,在所检测的样本中,荷斯坦种公牛和母牛CVM携带率分别为8.82%和5.71%,CVM基因频率分别为4.41%和2.85%。通过系谱追踪发现,CVM遗传缺陷的共同祖先是美国名牛＂Penstate Ivanhoe Star＂（USA.1441440,CV）。通过剔除CVM携带者公牛可以有效地控制CVM遗传缺陷的传播,但是,我国许多CVM携带者公牛冻精依然在商业化使用,所以有效地监控CVM携带者在奶牛群中的状况对CVM防控计划是有益的。     

荷斯坦奶牛脊椎畸形综合征焦磷酸测序检测方法建立与应用

为建立一种快速高通量的荷斯坦奶牛脊柱畸形综合征（complex vertebral malformation,CVM）的分子检测方法,根据CVM是由于SLC35A3基因第4外显子559位处发生G→T突变的遗传学基础,利用Assay Design SW软件设计了1对PCR引物和1条测序引物对三种不同基因型的基因材料进行测序分析,建立焦磷酸测序检测方法。对55份进境荷斯坦奶牛血样进行检测,发现有1例为隐性基因携带者,将PCR产物进行测序,其DNA序列与焦磷酸测序检测结果一致。研究表明该方法是一种有效的新方法,可用于荷斯坦奶牛脊椎畸形综合征的检测。     

Expression of Aquaporin Water Channels in Equine Endometrium is Differentially Regulated During the Oestrous Cycle and Early Pregnancy

The expression of 12 different aquaporin subtypes in equine endometrium was examined at the mRNA and protein level. Endometrial samples were obtained during anoestrus, oestrus, 8, and 14 days after ovulation in non‐pregnant mares, and 14 days after ovulation in pregnant mares. Quantitative PCR revealed a time‐dependent pattern for all aquaporin subtypes examined except for AQP10 and 12. AQP3, 5 and 7 showed highest mRNA abundance 8 days after ovulation, while AQP0 and 2 were most abundant at Day 14 of the cycle in non‐pregnant mares. At 14 days of pregnancy, AQP1, 4, 8, 9 and 11 displayed highest expression levels. Western blot analysis confirmed protein expression of AQP0, 2 and 5. Immunohistochemistry localized protein expression to luminal and glandular epithelial and stromal cells. AQP0 staining intensity was highest in samples obtained on Day 14 of the oestrous cycle. AQP2 immunoreactivity seemed to be stronger in samples collected 14 days after ovulation from non‐pregnant animals, in particular luminal epithelial staining. Samples collected 8 days after ovulation from cyclic animals were characterized by intense AQP5 staining of glandular epithelium, predominantly in the deeper glands. Progesterone treatment of anoestrous mares did not enhance expression of AQPs, indicating that factors other than progesterone are required for the up‐regulation of certain AQP subtypes during dioestrus. In conclusion, it seems that an equine‐specific collaboration of aquaporin subtypes contributes to changes in endometrial fluid content occurring throughout the oestrous cycle and contributes to endometrial receptivity during early pregnancy in the mare.     

荷斯坦牛脊椎畸形综合征分子诊断方法的建立与应用

脊椎畸形综合征(Complex Vertebral Malformation,CVM)是由常染色体上SLC35A3基因单碱基突变(G→T)引起的隐性遗传疾病,该基因隐性纯合(CV/CV)时奶牛致死,但CVM携带者表现正常,所以CVM携带者公牛可以通过人工授精技术传播CVM缺陷基因。本研究自行设计检测CVM特异性PCR引物,扩增长度为173bp,然后利用PCR-SSCP方法对186个公牛样品和140个母牛样品进行了检测分析,该方法简便快捷、准确率高,使用样品宽泛,适合大样本筛选。研究结果表明,在所检测的样本中,荷斯坦种公牛和母牛CVM携带率分别为11.3%和12.1%,并通过系谱追踪发现,CVM遗传缺陷的共同祖先是美国名牛"Penstate Ivanhoe Star"(USA.1441440,CV)。通过剔除CVM携带者公牛可以有效地控制CVM遗传缺陷,但是,我国许多CVM携带者公牛冻精依然在商业化使用,所以,有效地监控CVM携带者在奶牛群中的状况对CVM防控计划是有益的。     

Effect of Oestrous Cycle on the Oxidative Burst Activity of Blood Polymorphonuclear Leucocytes in Cows

Blood polymorphonuclear leucocyte (PMN) oxidative burst activity, plasma cortisol levels, and the total and differential white blood cells counts (WBC) of six cycled dairy cows were evaluated for a period of 24 days, three times a week; on Mondays, Wednesdays and Fridays. The PMN oxidative burst was indirectly evaluated by flow cytometry, measuring the intracellular oxidation of 2′,7′‐dichlorofluorescein diacetate to 2′,7′ dichlorofluorescein (DCF) by H₂O₂‐production. Results are pre‐sented as the mean fluorescence intensity (MFI) of DCF. Cow’s oestrous cycle was evaluated by following the plasma progesterone levels using a radioimmunoassay method. Levels of cortisol in the plasma were measured using a fluorimetric method. The oxidative burst activity of PMN, represented a maximum value (MFI = 117.6 ± 7.4) during the oestrous period. A fall was then observed, in which a steady state was observed during the lutheinic phase of the oestrous cycle, reaching the minimum value [MFI = 73.2 ± 11.2 (p ≤ 0.01)] on the days +8, +9 and +10. No significant variations were observed in the levels of cortisol, or in total and differential WBC, during the whole period. Nevertheless, as far as cortisol levels were concerned, a trend analogous to that of the oxidative burst activity was observed. Our results demonstrated that the oestrous cycle might influence directly, or indirectly, the immune system of cows, by altering the oxidative burst of PMN.     

Luteal Characteristics and Progesterone Production on Day 5 of the Bovine Oestrous Cycle

In this study we have examined luteal function in non-lactating and late lactation dairy cows on day 5 of the cycle, during the period of the post-ovulatory progesterone rise. Comparison of luteal progesterone content and in vitro synthetic capacity with circulating plasma progesterone demonstrated that circulating progesterone concentration is a function of total luteal activity rather than the activity of individual units of tissue. Incubation of luteal tissue in vitro demonstrated stimulatory activity of LH and IGF-I, and to a greater degree IGF-II, on luteal progesterone synthesis. Finally the study showed no effect of double ovulation on luteal function. Occurrence of double ovulation in 35% of animals was not associated with any difference in luteal function or plasma progesterone concentrations.     

Effect of Naloxone on Serum Luteinizing Hormone Concentration in Anovulatory Holstein Cows During the Early Postpartum Period

This experiment was conducted to investigate the effect of naloxone (NAL), an opioid receptor antagonist, on pituitary LH secretion in anovulatory Holstein cows during the early postpartum period when cows were expected to be in negative net energy balance. Twenty-three cows (11 primiparous) received either saline (n = 12) or 1 mg/kg BW NAL i.v. (n = 11) on Day 14 or 15 postpartum. Jugular blood samples were collected at 15-min intervals for 2 hr before and 2.5 hr after NAL or saline. All cows received 3 ug gonadotropin releasing hormone (GnRH) at 2.5 hr post-NAL or -saline and blood collection was continued for 1 hr. Mean serum progesterone concentration was 0.33 ± 0.2 ng/ml. Mean net energy balance for all cows was -5.5 ± 0.6 Mcal/day. Naloxone caused a transient increase (P < 0.05) in serum LH concentrations in both primi- and multiparous cows within 45 min after administration. In contrast, serum LH concentrations remained unchanged in saline-treated cows. GnRH increased (P < 0.05) LH and there was no effect of treatment. These results suggest that modulation of LH secretion, at least in part, may be mediated via endogenous opioids in dairy cows before first postpartum ovulation.     

提高奶牛受胎率的卵泡和黄体活动调控研究

在夏季和秋季对经产荷斯坦奶牛进行定时排卵-输精程序（TAI）处理并在输精后进行HCG处理（第5d和第10d分别肌注HCG2000IU／头），另于冬季单独对自然发情人工授精母牛进行输精后HCG处理。从各季节HCG处理组及其对照组中抽取部分奶牛，在输精后第0d（输精当天）、7d、14d、和21d分别采血，以测定孕酮和雌二醇水平。所有试验牛在输精后45-60d进行直肠妊娠检查以确定是否受胎。结果显示：夏季TAI＋HCG组、TAI组、对照组母牛输精后的受胎率差异不显著（p〉0．05），分别为26．67％（4／15）、16．67％（2／12）和10．71％（3／28）。秋季TAI＋HCG组、TAI组、对照组情期受胎率差异也不显著（p〉0．05），分别为：55．00％（11／20）、42．11％（8／19）和31．43％（11／35）。冬季HCG组和对照组的受胎率分别为63．16％（12／19）和45．00％（9／20）（P〉0．05）。在夏秋冬三个季节，输精后HCG处理相应显著提高第7d、14d和21d血液中孕酮水平（p〈0．05），但稍稍降低雌二醇水平（p〉0．05），显著降低E2／P比值（p〈0．05）。与受胎率的差异相关，秋冬季各组牛输精后血浆P水平高于（p〉0．05）或（极）显著高于（p〈0．01或p〈0．05）夏季相应组；血浆E2水平则稍高于（p〉0．05）夏季相应组。以上结果说明，TAI单独处理可以将受胎率提高约10～15个百分点；输精后单独HCG处理可以将爱胎率又提高约10-15个百分点，这主要是促进黄体功能的结果，而且提高的幅度在秋冬季要高于夏季。     

Effect of Meloxicam Treatment during Early Pregnancy in Holstein Heifers

We previously reported that administration of flunixin meglumine two times at a critical stage approaching pregnancy recognition associated with maintenance of the corpus luteum (CL) increased early embryo survival and pregnancy rate via an additive antiluteolytic effect with the conceptus [Vet Rec 160 (2007) 404]. In this study, the objective was to determine if a single administration of meloxicam, a non‐steroid anti‐inflammatory drug with a longer half‐life, could be used instead of flunixin meglumine. This would avoid repeated injections in heifers following insemination at a critical stage to increase pregnancy rate due to its inhibitory effect on prostaglandin F_2α synthesis. Eighty‐five, 15‐ to 18‐month‐old Holstein heifers were synchronized, and following insemination (day 0) heifers were assigned to receive subcutaneous meloxicam injection (0.5 mg/kg; n = 37) on the afternoon of day 15 or were untreated as a control (n = 48). Pregnancy rates were defined as the percentage of heifers inseminated that were diagnosed pregnant by ultrasound between days 31 and 38 after artificial insemination. Effect of the treatment on pregnancy rates was analysed by chi‐square test. Meloxicam treatment on day 15 after insemination dramatically decreased pregnancy rates in the heifers (52%; 25 of 48 in the control group vs 24.3%; 9 of 37 in the meloxicam‐treated group; p < 0.01). This result indicates that administration of meloxicam at the time associated with pregnancy recognition processes to maintain the CL was harmful to the pregnancy even though the drug is considered to be safe during pregnancy in cattle.     

Impact of Probing the Reproductive Tract During Early Pregnancy on Fertility of Beef Cows

This short communication reports the impact of endometrial biopsies, uterine flushings and follicular fluid aspiration procedures at day 6 post artificial insemination (AI) on pregnancy rates. In Experiment 1, cows were timed AI (TAI) and assigned to the following treatment groups: control (n = 37), uterine flushing (n = 35) and endometrial biopsy (n = 38). On day 30 post AI, pregnancy rates were 40.5%, 33% and 28.5%, respectively (p > 0.1). Pregnancy rate on day 60 was lower (p < 0.004) in flushed cows than in the controls. In Experiment 2, oestrus was detected and cows were assigned to flushing (n = 32) or biopsy (n = 33) treatments 6 days after AI, which resulted in pregnancy rates of 31% and 36%, respectively (p > 0.1). In Experiment 3, cows were, 6 days after TAI, randomly assigned to the following treatments: control (n = 84) or aspiration of the largest follicle (n = 73). Pregnancy rates on day 30 post AI were 63.5% for the control group and 53% for the aspirated group (p > 0.1). In conclusion, uterine flushing and endometrial biopsy negatively affect pregnancy rates, but neither procedure can be considered to be incompatible with pregnancy maintenance. Follicular aspiration during pregnancy does not interact with pregnancy success. The amount and quality of samples obtained are compatible with the use of cellular and molecular analysis of uterine variables from cows that failed or succeeded on maintaining pregnancy.     

褪黑素对荷斯坦奶牛妊娠率及生殖激素的影响

试验旨在探明皮下注射褪黑素（MT）对荷斯坦奶牛配种妊娠率及血清生殖激素的影响。用计步器法确定自然发情的首次配种荷斯坦奶牛150头,对其中70头进行颈部皮下肌内注射褪黑素30 mg,12 h后进行人工输精;选择170头产后首次配种的荷斯坦奶牛进行同期排卵-定时输精处理,其中90头荷斯坦奶牛最后一次注射促性腺激素释放激素（gonadotropin-releasing hormone,GnRH）的同时进行颈部皮下肌内注射褪黑素30 mg,16 h后进行人工输精。在进行二次配种的荷斯坦奶牛中选择153只进行皮下注射褪黑素。荷斯坦奶牛输精后20~35 d进行妊娠检查,详细记录首次配种妊娠母牛头数、二次配种妊娠母牛头数、产犊数。选取同期排卵-定时输精的荷斯坦奶牛25头,皮下注射褪黑素8 h后用放射免疫法检测其血清中褪黑素、促卵泡素（FSH）、促黄体素（LH）、雌二醇（E₂）的含量及35 d妊检时妊娠母牛血清中孕酮（P₄）含量。结果显示,与自然发情和同期排卵对照组相比,其对应的皮下注射褪黑素组荷斯坦奶牛的妊娠率及产犊率均显著提高（P<0.05）;皮下注射褪黑素组的双犊率显著提高（P<0.05）,首次配种妊娠率和产犊率均显著提高（P<0.05）,二次配种妊娠率和产犊率均差异不显著（P>0.05）。血清激素检测结果表明,与对照组相比,皮下注射褪黑素组血清中褪黑素、LH、E₂含量均显著增加（P<0.05）;35 d妊检时,皮下注射褪黑素的妊娠母牛P₄含量显著增加（P<0.05）。本试验结果表明,皮下注射褪黑素能够提高荷斯坦奶牛的妊娠率、产犊率及血清中LH、E₂和P₄含量,说明皮下注射褪黑素能够促进卵母细胞成熟和排卵,并提高配种妊娠率。